The Experimental Therapeutics Branch has long sought to elucidate molecular mechanisms contributing to the accelerated degeneration of nigral dopamine neurons in Parkinson's disease as an approach to discovering an effective neuroprotective therapy. The degenerative process appears to involve a defect in brain mitochondrial complex I in association with the activation of nuclear factor-kappaB (NF-kappaB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-kappaB and then the activation of caspase-3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone-induced nuclear translocation of NF-kappaB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-kappaB nuclear translocation and caspase-3 activation. PGA1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-kappaB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition. Branch research on novel approaches to the palliation of parkinsonism continues to build on our hypothesis that the nonphysiologic stimulation of striatal dopaminergic receptors, as a result of disease- or drug-related denervation or intermittent excitation, triggers adaptive responses in the basal ganglia which contribute to the appearance of parkinsonian symptoms and later to the dyskinesias and other alterations in motor response associated with dopaminergic therapy. Our results suggest that these altered responses involve activation of signal transduction cascades in striatal medium spiny neurons linking dopaminergic to coexpressed ionotropic glutamatergic receptors of the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) classes. As their phosphorylation state changes, their synaptic efficacy rises, thus enhancing cortical glutamatergic input which in turn modifies striatal output in ways that compromise motor behavior. Since protein kinase C (PKC)-mediated phosphorylation regulates glutamatergic receptors of the AMPA subtype and has been linked to several forms of behavioral plasticity, activation of PKC signaling in striatal spiny neurons may also contribute to the motor plasticity changes associated with chronic levodopa therapy. To evaluate this possibility, we augmented PKC signaling by using Herpes Simplex Virus type 1 vectors (pHSVpkcDelta) to directly transfer the catalytic domain of the PKCbetaII gene into striatal neurons of parkinsonian rats. Microinjection of pHSVpkcDelta vectors lead to the persistent expression of PkcDelta in medium spiny neurons together with an increase in serine 831 phosphorylation on AMPA receptor GluR1 subunits and hastened the appearance of the shortened response duration produced by chronic levodopa treatment. The intrastriatal injection of a PKC inhibitor attenuated both the increased GluR1 phosphorylation and the accelerated onset of the levodopa-induced response modifications. However, in rats that received levodopa treatment for 21 days without the gene transfer, intrastriatal NPC-15437 had no effect on the response shortening or on GluR1 S831 phosphorylation. The results suggest that an increase in PKC-mediated signaling, including phosphorylation of AMPA receptors on striatal spiny neurons may be sufficient to promote the initial appearance, but not necessary for the ultimate expression, of the levodopa-induced motor response changes occurring in a rodent model of the human motor complication syndrome. Activation of cAMP responsive element binding protein (CREB) has been increasingly implicated in the maintenance of long-term memory. To evaluate the possibility that CREB might participate in molecular mechanisms underlying the persisting alterations in motor response occurring with levodopa treatment of parkinsonian patients, we evaluated the time course of these changes in relation to the activation of striatal CREB in 6-hydroxydopamine lesioned rats. Three weeks of twice-daily levodopa treatment reduced the duration of the motor response to acute levodopa challenge, which lasted about 5 weeks after withdrawal of chronic levodopa therapy. This shortened response duration, resembling human wearing-off fluctuations, was associated with an increase in Ser-133 phosphorylated CREB in spiny neurons in dorsolateral striatum in response to acute dopaminomimetic challenge. The time course of changes in CREB phosphorylation correlated with the time course of changes in motor behavior after cessation of chronic levodopa therapy. Both the altered motor response duration and the degree of CREB phosphorylation were attenuated by the intrastriatal administration of CREB antisense or protein kinase A inhibitor Rp-cAMPS. The results suggest that region-specific Ser-133 CREB phosphorylation in D1 receptor containing spiny neurons does indeed contribute to the persistence of the motor response alterations produced by intermittent stimulation of striatal dopaminergic receptors. During the past year Branch investigators found that adenosine A2a receptors signal via kinases whose aberrant activation contributes to the appearance of parkinsonian signs following dopaminergic denervation and to the motor response complications produced by dopaminomimetic therapy. To assess the ability of A2a receptor blockade to normalize certain of these kinases and thus benefit motor dysfunction, the palliative and prophylactic effects of the selective antagonist, KW-6002, were first evaluated in rodent and primate models. In hemiparkinsonian rats, KW-6002 reversed the intermittent levodopa treatment-induced, protein kinase A-mediated, hyperphosphorylation of striatal AMPA receptor GluR1 S845 residues as well as the concomitant shortening in motor response duration. In MPTP lesioned monkeys, coadministration of KW-6002 with daily apomorphine injections acted prophylactically to prevent dyskinesia onset. These preclinical observations guided the design of a randomized, controlled, proof-of-concept study of the A2a antagonist in moderately advanced parkinsonian patients. While KW-6002 alone or in combination with a steady-state intravenous infusion of optimal-dose levodopa had no effect on parkinsonian severity, the drug potentiated the antiparkinsonian response to low dose levodopa with less dyskinesias than produced by optimal dose levodopa alone. KW-6002 also safely prolonged the efficacy half-time of levodopa. These results suggest that drugs capable of selectively blocking adenosine A2a receptors could confer therapeutic benefit to levodopa treated parkinsonian patients and warrant further evaluation in Phase II studies. They also illustrate a strategy for successfully bridging a novel approach to PD therapy from an evolving research concept to pivotal clinical trials.